Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that plays a critical role in regulating mammalian oxygen homeostasis (Bunn, et al (1996) Physiol Rev 76(3), 839–85; Semenza, (1999) Annu Rev Cell Dev Biol 15, 551–78; Wenger, (2000) J Exp Biol 203 Pt 8, 1253–63). Adaptation to changes in oxygen tension involves a variety of developmental, physiological, and pathophysiological processes including embryonic development, angiogenesis, cerebral and myocardial ischemia, and tumorigenesis (Semenza, (2000) Genes Dev 14(16), 1983–91; Semenza, (2002) Trends Mol Med 8(4), S62–7) HIF-1 consists of HIF-1α and HIF-1β subunits, both of which belong to the basic helix-loop-helix Per-AhR-Sim family (Wang, et al (1995) J Biol Chem 270(3), 1230–7; Wang, et al (1995) Proc Natl Acad Sci USA 92(12), 5510–4). Under hypoxia, HIF-1 becomes activated and up-regulates target genes such as erythropoietin, vascular endothelial growth factor, glucose transporter, and glycolytic enzymes (Semenza, (1999) Annu Rev Cell Dev Biol 15, 551–78).
HIF-1 activation is regulated primarily by the accumulation of HIF-1 alpha protein (Huang, et al H. F. (1996) J Biol Chem 271(50), 32253–9). Both HIF-1 alpha and HIF-1 beta genes are constitutively expressed in many cell lines examined (Huang, et al (1996) J Biol Chem 271(50), 32253–9; Gradin, et al (1996) Mol Cell Biol 16(10), 5221–31; Wood, et al (1996) J Biol Chem 271(25), 15117–23; Kallio, et al (1997) Proc Natl Acad Sci USA 94(11), 5667–72), whereas HIF-1α protein is constantly degraded under normoxia by the ubiquitin-proteasome pathway (Huang, et al (1998) Proc Natl Acad Sci USA 95(14), 7987–92; Kallio, et al (1999) J Biol Chem 274(10), 6519–25). The degradation is controlled by a unique oxygen-dependent degradation domain (ODD) consisting of ˜200 amino acids within HIF-1α (Huang, et al (1998) Proc Natl Acad Sci USA 95(14), 7987–92). Deletion of the entire ODD gave rise to a stable HIF-1α, capable of heterodimerization, DNA-binding, and transactivation in cell culture systems. Consistently, the ODD-deleted HIF-1α (but not the full-length), when transgenically expressed in the mouse epidermis, activated HIF-1 target genes, thereby resulting in epidermal hypervascularity (Elson, et al (2001) Genes Dev 15(19), 2520–32), providing compelling evidence that a stable HIF-1α, irrespective of hypoxic signal, is sufficient for transcriptional activation in animal models.
Studies on the mechanisms underlying HIF-1α degradation, Maxwell et al. first reported that the tumor suppressor protein, VHL, targets HIF-1α for oxygen-dependent proteolysis in an iron-dependent way (Maxwell, et al (1999) Nature 399(6733), 271–5). Inactivation of the VHL (von Hippel-Lindau) gene is linked to the development of the VHL disease, a hereditary human cancer syndrome characterized by the predisposition to develop highly angiogenic tumors (Kaelin, et al (1998) Trends Genet 14(10), 423–6). The VHL protein is in a multiprotein complex with elongin B, elongin C, and Cul2, which share sequence similarity with the Skp1 and Cdc53 components of the SCF ubiquitin ligase (Duan, et al (1995) Science 269(5229), 1402–6; Kibel, et al (1995) Science 269(5229), 1444–6; Pause, et al (1997) Proc Natl Acad Sci USA 94(6), 2156–61; Stebbins, et al (1999) Science 284(5413), 455–61). Furthermore, VHL is associated with Rbx1 or ROC1 (Kamura, et al (1999) Science 284(5414), 657–61), a potent SCF ubiquitin ligase activator that facilitates degradation of substrate proteins by recruiting a ubiquitin-conjugating enzyme to the complex (Ohta, et al (1999) Mol Cell 3(4), 535–41; Tan, et al (1999) Mol Cell 3(4), 527–33). All these observations have led to the hypothesis that the VHL complex functions as an E3 ubiquitin ligase for HIF-1α polyubiquitination (Kamura, et al (2000) Proc Natl Acad Sci USA 97(19), 10430–5) by specifically targeting the ODD of HIF-1α (Cockman, et al (2000) J Biol Chem 275(33), 25733–25741; Ohh, et al (2000) Nat Cell Biol 2(7), 423–427; Tanimoto, et al (2000) Embo J 19(16), 4298–4309).
VHL binding requires specific recognition of hydroxylated Pro-564 of HIF-1α (Ivan, et al (2001) Science 292(5516), 464–8.; JAAkkola, et al (2001) Science 292(5516), 468–72.; Yu, et al (2001) Proc Natl Acad Sci USA 98(17), 9630–5). Proline hydroxylation, catalyzed by a conserved family of prolyl-4-hydroxylases, relies on molecular oxygen and iron (Epstein, et al (2001) Cell 107(1), 43–54.; Bruick, R. K., and McKnight, S. L. (2001) Science 294(5545), 1337–40), indicating these enzymes act as oxygen sensors. Moreover, recent studies of HIF1α-VHL complexes provide a structural basis for VHL recognition of hydroxyproline in HIF-1α (Hon, et al (2002) Nature 417(6892), 975–8.; Min, et al (2002) Science 296(5574), 1886–9), suggesting a central role for proline hydroxylation in oxygen signaling. Interestingly, Pro-402 of HIF-1α is also subjected to hydroxylation and, in turn, targeted by the VHL E3 ubiquitin ligase for HIF-1α ubiquitination (Masson, et al (2001) Embo J 20(18), 5197–206).